Abstract Methamphetamine (METH), a potent addictive psychostimulant, is one of the most commonly abused drugs in the United States. METH abuse is highly prevalent in HIV-infected individuals, which presents unique challenges for HIV prevention and treatment. Given the overlap impact of METH use and HIV on neuronal damage in the central nervous system (CNS), it becomes urgent to understand the role of interplays between METH and HIV in the pathogenesis of HIV-associated neurocognitive disorders (HAND). However, studies of HAND have been hampered by difficulties in collecting live brain cells from autopsy or biopsy of HIV patients. Recent success in generating microglia and cerebral organoids from human induced pluripotent stem cells (iPSCs) now offers a great opportunity to investigate the impact of METH and/or HIV on the CNS. The overall objective of this project is to validate and establish a clinically relevant in vitro brain model for NeuroAIDS research in the context of drug abuse. The project will be carried out by two P.I.s who have the complementary expertise and experience in the proposed studies. Dr. Ho has been working on the impact of abused drugs (METH and opioids) on peripheral/CNS innate immunity and HIV infection of macrophages/microglia since 1999. Dr. Hu?s research focuses on neurogenesis, iPSC generation, inflammasomes, CRISPR/Cas HIV eradication and HAND. Their recent collaborative work has successfully generated and characterized the human iPSC-derived microglia-containing cerebral organoids (MCOs) which express the major CNS cell types: neural stem/progenitor cells, neurons, astrocytes, and microglia. More importantly, these MCOs could be infected by HIV. Based on these important findings, we propose three specific aims: Aim 1. To study dynamic HIV infection of human iPSC-derived MCOs and the impact of METH on HIV infection of MCOs; Aim 2. To determine HIV-infected cell types and the primary target/reservoir cells of HIV in MCOs; Aim 3. To study the impact of HIV infection on the development and neuronal circuitry in MCOs. Successful completion of this project should provide an in vitro brain model to study the roles of HIV infection and drugs of abuse in the pathogenesis of NeuroAIDS.